The present invention relates to a process for the preparation of xcex1-amylase, useful for starch saccharification from a novel plant source Tinospora cordifolia Miers belonging to Menispermaceae group of plants. The present invention particularly relates to a process for the preparation of a novel xcex1-amylase, which saccharify starch mainly into maltose and glucose.
Starch degrading xcex1-amylase enzyme have large numbers of biotechnological applications e.g. in the productions of syrups containing oligosaccharides, maltose and glucose from corn and other starchy materials, fermentable carbohydrate for ethanol production by saccharification of corn or other starchy materials and as digestive enzyme in pharmaceutical preparations, as thinning agent in lowering viscosity of commercial starch preparations, improving enzyme in bread making and in many other applications. The traditional process of acid catalyzed saccharification of starch into syrup or fermentable sugars has largely been replaced for many advantageous reasons by the enzymatic processes using xcex1-amylase. References may be made to H. C. Barford, Cereal Foods World 21, 588, 1976 and L. A. Underkofler, L. J. Denault and E. F. Hon, Die Starke 17, 179, 1965.
The enzyme xcex1-amylase (xcex1-1,4 D-glucan glucanohydrolase Enzyme commission number 3.2.1.1) is widely produced by different microorganisms and is also present in some cereals. It hydrolyses xcex1-1,4-glucosidic linkages present in amylose, amylopectin and glycogen in an endo-fashion. But it does not hydrolyze xcex1-1,6-glucosidic branch point present in amylopectin. Saccharification of amylose and amylopectin by xcex1-amylase produces, maltose, maltotriose and glucose and a series of branched xcex1-limit dextrins respectively. The amount and nature of xcex1-limit dextrins varied with the nature of xcex1-amylase obtained from different sources.
The enzyme produced by microorganism may be saccharifying or liquefying in nature. Saccharifying enzyme produces more reducing sugar from starch than the liquefying enzyme, but later lower viscosity of starch more quickly than the former enzyme. Bacillus subtilis var amylosaccharficus, Bacillus subtilis Marburg, Bacillus subtilis Natto are potential producers of saccharifying xcex1-amylase. Reference may be made to H. Matsuzaki, K. Tamane, K. Yamaguchi, Y. Naguta and B. Maru Biochimica Biophysica Acta 365, 235, 1974. Bacillus amyloliqurefaciens produces large amount of liquefying xcex1-amylase. Reference may be made to N. G. Welkar and L. L. Campbell. Journal of Bacteriology 94, 1131, 1967, Saccharifying xcex1-amylase is immunologically different from liquefying enzyme and in having maltase activity. Reference may be made to H. Yoshida, K. Hiromi and S. Ono. Journal of Biochemistry, Tokyo, 62, 439, 1967. Saccharifying xcex1-amylase has tremendous uses in the production of syrup and fermentable carbohydrate from starchy raw materials and in medicine. Grain starch as carbon source, is used to the extent of 50% of total substrate for ethanol production. Liquefying amylase is used for quickly reducing viscosity of starch solution. Germinating cereals also contain saccharifying xcex1-amylase. Traditional starch hydrolysis is usually conducted using barley malt. Malt is obtained mainly from barley, sometimes also from wheat and oats. The cereals are allowed to germinate for a limited period of time and then dried under suitable condition to terminate growth of embryo. The ground powder of the dried cereals is used as the source of enzyme. In beer preparation, malt is used both as source of enzyme and source of carbohydrate for fermentation. In the preparation of whisky and other liquors, gelatinized starch is saccharified by malt enzyme and the hydrolysed product is fermented.
As an alternative to barley malt, saccharifying xcex1-amylase produced by different fungi are also largely used. Mold bran containing growth of Aspergillus oryzae is traditionally used for starch hydrolysis. Reference may be made to J. Ziffer and M. C. Losit. Biotechnology Letters 4, 573, 1982. Submerged fermentation process for the production of saccharifying xcex1-amylase was also developed and enzymes from Mucor touxii, Mucor boulard, Rizopus delemer etc. were used for starch saccharification.
However, the production of saccharifying xcex1-amylase by malting barley is a highly technical exercise, which depends on the use of selected varieties of cereals and on the malting technique, which was developed as an art over hundreds of year. On the other hand, bacterial and fungal enzymes have restricted use in food preparations. In the industrial production of fungal enzyme, frequent allergic outbreaks were reported time to time, possibly due to spreading of fungal spores in the environment.
The main object of the present invention is to provide a process for the preparation of xcex1-amylase useful for starch saccharification from a novel plant source Tinospora cordifolia Miers belonging to Menispermaceae group of plant.
Another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the enzyme preparation does not require presence of calcium ion for optimum activity.
Still another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the saccharifying enzyme has xcex1-glucosidase activity which hydrolyses maltose.
Yet another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the production of enzyme does not require any controlled environmental conditions like malting or growth of microorganism under defined physicochemical conditions.
One more object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the crude enzyme preparation contains mostly xcex1-amylase protein with little non-amylase contaminating protein.
One another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the enzyme preparation become homogeneous protein in gel electrophoresis in presence of sodium dodecyl sulphate by a single step process involving ion exchange chromatography.
Another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the enzyme preparation under suitable conditions could digest soluble starch until the degree of polymerization is between 2 and 3.
Still another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the enzyme preparation under suitable conditions could digest soluble starch to generate up to 20% free glucose.
Yet another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the starting material used as the source enzyme is a plant which has been known to be edible to humans for a longtime.
One more object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the plant used as starting material is not a vegetable or fodder having alternative demand.
One another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the plant used as starting material grows wild throughout the whole seasons.
Another object of the present invention is to provide a process for the preparation of xcex1-amylase wherein the plant used as starting material could grow from any part of the mature stem under proper growth supporting environments.
The present invention provides a process for the preparation of xcex1-amylase useful for starch saccharification from a novel plant source Tinospora cordifolia Miers belonging to Menispermaceae group of plant.
Accordingly, the present invention provides a process for the preparation of xcex1-amylase useful for starch saccharification, which includes blending cut pieces of Tinospora codifolia Miers stem in an aqueous medium at a pH of 4.5 to 8.0 and a temperature between 0xc2x0-50xc2x0 C., separating the aqueous phase by a known process of filtration, concentrating the extract by a known process of enzyme concentration, isolating the pure xcex1-amylase by conventional protein purification methods.
In an embodiment of the present invention, the extraction medium used is water, acidulated water or mild alkaline water of pH from 4.5 to 8.0, buffer of pH 4.5 to 8.0, or water containing 0-10% (v/v) organic solvents such as acetone or ethanol.
In a preferred embodiment of the present invention, the pH value of the medium is between 5 and 6.
In another preferred embodiment of the present invention, the temperature of the liquid medium is maintained at 30xc2x0 C.
In another embodiment of the present invention, the reagents used for the precipitation of enzyme are selected from the group consisting of acetone, ethanol, polyethylene glycol, ammonium sulphate, and sodium sulphate.
In still another embodiment of the present invention, process used for the concentration of enzyme solution is selected from the group comprising of ultrafiltration, lypophilisation, vacuum distillation and aqueous two-phase systems containing dextran or sodium chloride etc.
In yet another embodiment of the present invention, purification of xcex1-amylase is done by ion exchangers or gel filtration method.
In another embodiment of the present invention, the ion exchangers used for the purification of xcex1-amylase are weak anion exchangers or weak cation exchangers.
In still another embodiment of the present invention, weak anion exchangers used for the purification of xcex1-amylase are one or more of diethylaminoethyl (DEAE)-sephadex, DEAE-cellulose, DEAE-sepharose, DEAE-sephacel, DEAE-cellulose, Epichlorohydrin triethanolamine-cellulose, Diethyl-[2-hydroxypropylamino ethyl (QAE)]-Sephadex, QAE-cellulose and DEAE-Trisamyl.
In yet another embodiment of the present invention, cation exchangers used for the purification of xcex1-amylase are one or more of carboxymethyl (CM)-Sephadex, CM-agarose, CM-cellulose, Sulfopropyl- (SP)-Sephadex, SP-sepharose and Cellulose phosphate sulfoxyethyl-cellulose.
In one more embodiment of the present invention, gel filter media used for the purification of xcex1-amylase are one or more of sephadex, sephacryl, sepharose, superose, tyopearls, ultrogel and beaded cellulose.
In one another embodiment of the present invention, production of enzymes does not require any controlled environmental conditions such as malting or growth of microorganism under defined physiochemical conditions.
In another embodiment of the present invention, production of enzymes does not require presence of calcium ion.
In still another embodiment of the present invention, the crude enzyme contains mostly xcex1-amylase protein with little non-amylase contaminating protein.
In yet another embodiment of the present invention, the enzyme preparation become homogeneous protein gel electrophoresis in the presence of sodium dodecyl sulphate by a single step process involving ion exchange chromatography.
In one more embodiment of the present invention, the enzyme prepared could digest soluble starch until the degree of polymerization is between 2 and 3.
In another embodiment of the present invention, the enzyme prepared could digest soluble starch to generate up to 20% free gluose.
In another embodiment of the present invention, the extract may be used as a direct source of saccharifying xcex1-amylase.
In still another embodiment of the present invention, the enzyme has highest activity between temperatures of 60xc2x0 and 65xc2x0 C. and at a pH of 6.0xc2x10.2.
In yet another embodiment of the present invention, the activity was found to be stable up to a temperature of 60xc2x0 C. and a pH of 7.5.
In another embodiment of the present invention, total recovery of xcex1-amylase activity was about 980 units.
In one another embodiment of the present invention, the enzyme gave a single protein band of 43-kilodalton weight in SDS-PAGE.
In another embodiment of the present invention, total recovery of xcex1-amylase activity was 980 units.
The enzyme is obtained from the plant Tinospora cordifolia Miers, is a known medicinal plant called by different names like Guruchi, Amritballi, Gulancha, Tippatigi, Sindi, Guthabael, Golo etc in different regions of India and abroad. It grows throughout the tropical India, Maynamar, and Srilanka. Reference may be made to Indian Medicinal plants, Volume 1, Edited by Lt.Col. K. R Kirtikar, Major B D Basu and An I.C.S, Revised by E Blatter, J. F Caius and K s Mhaskar, pp 77-80 Published by Bishen Sing Mahendra Pal Sing, Cannught Place, Dehradunn-248001, India (1998).
The mature plants grown wild in different fields were collected and sorted to eliminate infected part, if any. Stem parts having a thickness of more than 2 millimeters were freed from leaves and thoroughly washed with water. These stems were cut into thin slices of 1-3 mm thickness by sharp knife. The cut pieces were immersed immediately in water or a buffer of pH from 4.5 to 8.0, preferably of pH 5.0 to 6.0. Volume of liquid was from 100 ml to 500 ml per 100 gram of green stem, preferably 200 ml per 100-gram plant. Temperature of the extracting liquid was 0-50xc2x0 C., preferably below 30xc2x0 C. The mixture was blended in a Waring blender to obtain a paste of the biomass. The mass was squeezed through a nylon cloth to obtain a greenish liquid free from cellulosic fiber. The residue was resuspended in the same volume of water and re-blended for 1 minute and similarly passed through nylon cloth. The total filtered liquid was kept for 4 hours at room temperature when precipitation of a white mass takes place. The clear liquid was filtered out under suction. The clear liquid could be used as the source of saccharifying xcex1-amylase enzyme, which contains xcex1-amylase, as the major constituent protein along with some minor proteins in the solution. The saccharifying enzyme was purified either by gel filtration medium capable of separating proteins of molecular weight between 40-50 kilodalton, or by absorption of weak amino exchanger, preferably DEAE-cellulose at pH 5-6.0 and elution under 0-1M salt gradient or by passing through a weak cation exchanger, preferably CM-cellulose and collecting the extract. The enzyme purified by any one of the process is a homogeneous protein as found by sodium dedecyl sulphate-polyacrylamide gel electrophoresis. The molecular weight is in the range of 40-45 kilodalton.
The enzyme activity is determined by estimating the amount of reducing sugar liberated from the substrate by dinitrosalicylic acid method. Reference may be made to J. B. Sumner and G. Sumner in xe2x80x9cLaboratory Experiments in Chemistryxe2x80x9d Academic Press, New York, pp.38, 1949. Unit of enzyme activity was taken as the amount of enzyme, which could liberate one micromole of maltose equivalent per minute in buffer of pH 6.0 at 50xc2x0 C. The xcex1-glucosidase activity of the enzyme preparation before purification was assayed with p-nitrophenyl-xcex1-D-glucoside as substrate. Reference may be made to S. Sengupta and S. Sengupta. Canadian Journal of Microbiology 36 (9), 617, 1990. One unit of enzyme activity was taken as the amount of enzyme which could liberate one micromole of p-nitrophenol from p-nitrophenyl-xcex1-D-glucoside in the reaction mixture incubated at 50xc2x0 C. for 30 minutes. The xcex1-amylase activity of the purified or of the unpurified extract active on starch in the pH range of 4 to 7.5, optimally at 6.0, at temperature between 20-70xc2x0 C., optimally at 60xc2x0 C., with velocity maximum (Vm) value on Michaelis constant (Km) value being 34.8 unit per milligram of protein and 3.75 mg soluble potato starch per milliliter in the incubation mixture. The enzyme activity does not require any calcium ion for optimum activity. The enzyme, under optimum reaction condition, for example pH temperature, enzyme-substrate ratio (Km) digest soluble potato starch to reducing sugar of degree of polymerization between 2-3. The micelles constant (Km) value of enzyme with soluble potato starch as substrate varies from 3.5-4.0 mg soluble starch/milliliter and the corresponding velocity maximum value ranged from 30-40 xcexcmoles of reducing group per minute per milligram of protein at the incubation temperature of 50xc2x0 C.